CHARACTERIZATION OF A CHIMERIC PLASMINOGEN-ACTIVATOR CONSISTING OF A SINGLE-CHAIN FV FRAGMENT DERIVED FROM A FIBRIN FRAGMENT D-DIMER-SPECIFIC ANTIBODY AND A TRUNCATED SINGLE-CHAIN UROKINASE

An M(r) 57,000 single-chain chimeric plasminogen activator, K12G0S32, Consisting of a variable region fragment (Fv) derived from the fibrin fragment D-dimer-specific monoclonal antibody MA-15C5 and of a 33-kDa (amino acids Ala132 to Leu411) recombinant single-chain urokinase-type plasminogen activator (rscu-PA-33k) was studied. K12G0S32, Secreted by infected Spodoptera frugiperda insect cells at a rate of 1.5-mu-g/10(6) cells/48 h, was purified to homogeneity by ion-exchange chromatography and gel filtration. It was obtained essentially as a single-chain molecule with a K(a) 5.5 x 10(9) M-1 for immobilized fragment D-dimer, similar to that of MA-15C5. The specific activity of both its single-chain and two-chain forms on fibrin plates was 100,000 IU/mg of urokinase-type plasminogen activator (u-PA) equivalent. Activation of plasminogen by two-chain K12G0S32 obeyed Michaelis-Menten kinetics with K(m) = 2.9 +/- 0.6-mu-M and a k2 = 3.7 +/- 0.6 s-1 (mean +/- S.D.; n = 3), as compared to K(m) = 12-mu-M and k2 = 4.8 s-1 for rtcu-PA-32k (recombinant low M(r) two-chain u-PA consisting of amino acids Leu144 to Leu411). Single-chain K12G0S32 induced a dose- and time-dependent lysis of a I-125-fibrin-labeled human plasma clot immersed in citrated human plasma; 50% lysis in 2 h was obtained with 0.70 +/- 0.07-mu-g/ml (mean +/- S.D.; n = 5), as compared with 8.8 +/- 0.1-mu-g/ml for rscu-PA-32k (recombinant low M(r) single-chain u-PA consisting of amino acids Leul44 to Leu411) (mean +/- S.D.; n = 3). With two-chain K12G0S32, 50% clot lysis in 2 h required 0.25 +/- 0.03-mu-g/ml (mean +/- S.D.; n = 3), as compared with only 0.62 +/- 0.04-mu-g/ml (mean +/- S.D.; n = 2) for rtcu-PA-32k. These results indicate that low M(r) single-chain u-PA can be targeted to a fibrin clot with a single-chain Fv fragment of a fibrin-specific antibody, resulting in a 13-fold increase of the fibrinolytic potency of the single-chain form and a 2.5-fold increase of the potency of the two-chain form.